I. Field of the Invention
This invention relates to selectable and reporter genes (sometimes referred to as dominant selectable marker genes) for use during the genetic engineering of plant cells to permit selection, detection and analysis of transformed plant cells and plants. More particularly, the invention relates to such genes, plasmids and vectors containing such genes, procedures for selecting transformed cells and plants containing such genes and procedures for detecting the presence of such genes in transformed plants.
II. Description of the Prior Art
Currently there are very few genes available for use during the genetic engineering of plants that can be used effectively for the selection, detection and analysis of transformed plants and plant cells (Walden et. al, Methods in Molecular and Cellular Biology 1: 175-194, 1990; the disclosure of which is incorporated herein by reference). This is an impediment to rapid and effective manipulation of genetic material in plants and makes the application of this technology more difficult than for other types of cells and organisms, i.e. animal cells, insect cells, microorganisms, and the like.
Most of the genes which are currently available for use in the selection of transformed plants or plant cells are either antibiotic- or herbicide-resistant genes, which tend to vary in their effectiveness in different plant species or to adversely interfere with the plant regeneration process. Additionally, the products of such genes are, in many cases, enzymes which cannot easily be detected or asseyed (Jefferson, Genetic Engineering: Principles and Methods 10: 247-263, 1988 Palenum Press).
Accordingly, the lack of effective single, selectable/reporter genes applicable to the plant kingdom has made it necessary to develop complex multigene transformation systems based on selectable antibiotic or herbicide resistance genes used in combination with other reporter genes, the products of which can be detected or assayed.
Currently, there are three general approaches used for providing selectability and detectability during genetic transformation of plants. In a first approach, a single, selectable gene, conferring antibiotic or herbicide resistance, is introduced to plant cells at the time of transformation. The gene confers resistance to a selecting agent, such as the antibiotic neomycin (Herrera-Estrella et. al., European Molecular Biology Organization Journal &lt;hereinafter "EMBO J."&gt; 2: 987-995, 1983) or the herbicide phosphinothricin (DeBlock et.al., EMBO J. 6: 2513-2518, 1987).
Shortly after transformation, cells are exposed to the selection agent which kills nontransformed cells. In some applications, the products of these genes are not easily assayed (Reiss et. al., Gene 30: 211-218, 1984).
In a second approach, two separate genes are introduced into plant cells at the time of transformation. One of the genes is a selectable gene, e.g. the gene expressing neomycin phosphotransferase-II (NPT-II), used to confer resistance to selection agents, such as the antibiotic kanamycin (Herrera-Estretta et. al., supra). The other of the genes is a reporter gene, e.g. .beta.-glucuronidase (GUS) gene. This enzyme is relatively easy to assay for and produces an indigo colored product when incubated with the 5-bromo-4-chloro-3-indolyl glucuronidase [XGluc] (Jefferson et. al., EMBO J. 6: 3201-3207, 1987).
In the third approach, two separate genes such as NPT-II and GUS are fused together to produce a single bifunctional gene product referred to as a fusion gene (NPT-II/GUS), which confers resistance to a selection agent such as kanamycin and also confers the ability to produce a characteristic color reaction when incubated with XGluc (Datla et al., Gene 101: 239-246, 1991).
Unfortunately, the use of either multigene or fusion gene systems which depend on antibiotic resistance or herbicide resistance as the selectable component has raised concerns regarding potential undesirable effects in the agricultural industry and on environmental grounds (Gressel, TIBTECH 10: 382, 1992; Bryant & Leather, TIBTECH 10: 274-275, 1992; and Flavell et. al., Bio/Technology 10: 141-144, 1992).
There is therefore a need for an improved system allowing for the selection, detection and analysis of transformed plant cells.